The present invention relates to cell culture media and compositions for use in making such media.
Cell culture media are used for the culture of a wide range of cell types under varying circumstances and for varying purposes, which may or may not involve the division and multiplication of the cells. The term xe2x80x9ccell culture mediumxe2x80x9d is used herein to refer to any medium in which cells are maintained in vitro in an active and viable state.
As is well understood in this art, the culture of cells requires the supply to the cells of the materials which they need for the maintenance of viability and, if desired, for the production of cell products and/or the multiplication of the cells. Whilst many of these materials may be supplied in purified or synthetic form, serum has traditionally been required for the supply of others. The simpler materials required by cells in culture may be formulated into a basal cell culture medium typically containing inorganic salts, amino acids, vitamins, and various other additives. A number of formulations of such basal media have been proposed. In such basal media without serum supplementation however, cells quickly die. Serum contains a number of biochemical entities that the cells need for survival in such basal media. Some of these entities protect the cells against toxic impurities in the basal media, some of which may be products of the cells themselves, and others serve to present iron and trace metals to the cells in a way the cells can use. The addition of serum can produce a well functioning medium for many different cell types, but serum brings with it severe disadvantages.
These have driven attempts to devise successful cell culture media that are serum-free. These disadvantages include the following:
serum is a non-physiological fluid for cells, due to many differences in composition compared to the interstitial tissue fluid and also when compared to plasma;
serum has an inflammatory and transforming effect on cells inducing hyperstimulation and other phenomena due to its content of products released from blood platelets and leukocytes;
antibodies and plasma factors produce strong or destructive interference in scientific experiments and in biotechnological production. For instance, the production of purified monoclonal antibodies is made much more difficult;
since the blood stream constitutes a transport system for nutrients and also for waste products, all kinds of waste products from the body""s food intake and metabolism will be presented to the cultured cells, possibly with slaughter house contaminants;
serum is subjected to a heat treatment to inactivate lytic components which also has a denaturing effect on several serum proteins;
serum is not a reproducible and fixed material. Significant batch to batch variations interfere with the reproducibility of culture processes;
serum may contain known and unknown undesirable materials including viral or prion-type pathogens; and
serum may be expensive and difficult to acquire.
Lastly, while serum-containing media have been especially successful for cells of mesodermal origin, cells originating from embryonic ectoderm or endoderm have proved most difficult to keep in good condition in serum-supplemented basal media. This is also true for gametes, both egg cells and sperm cells.
A major advance in the formulation of cell culture media eliminating the presence of serum was described by us in U.S. Pat. No. 5,045,454. There we described an additive to basal media comprising EDTA in combination with citrate buffer to complex iron and prevent its precipitation and aurintricarboxylic acid to present the iron effectively to the cells. The present invention provides a number of distinct and separately useful improvements to the media previously available, each of which is particularly useful by itself or in combination with one or more of the others in improving the media described in U.S. Pat. No. 5,045,454.
The inclusion of lipids in cell culture media is a long standing problem. Lipids which it is desired to include have a variety of differing functions. They include long chain fatty acids, fat soluble vitamins and sterols such as cholesterol. The direct addition of lipids is not practical because of their low solubility. Conventionally in serum containing media, the lipids are added to the medium in the serum, wherein the lipids are carried as soluble lipoproteins. In serum free media, lipids can be carried by albumen, but it is of course desirable to avoid the use of albumen also. A number of publications including xe2x80x98Nutritional and Hormonal Requirements of Mammalian Cells in Culturexe2x80x99 D. Barnes, Wld Rev. Nutr. Diet., vol. 45 pp 167-197 describe the provision of lipids in cell culture via the inclusion of liposomes formed using phospholipids such as are found in such sources as cod liver oil (WO-A-8901027 and others). For convenience, the cholesterol which is to be included in the medium may be added with the lipids in such liposomes.
Such methods involve the use of poorly characterised materials such as cod liver oil, serving as the source of the phospholipids needed for producing the liposomes or microemulsions needed. This again is an undesirable feature of such media. There remains a need for a more acceptable method of including cholesterol in the media.
U.S. Pat. No. 4,533,637 describes the formation of inclusion complexes between lipids and cyclodextrins. We have found that lipid fatty acids and other lipids complexed with cyclodextrins do not function well in cell culturexe2x80x94in part due to low stability in solution.
xe2x80x98Biochemistry-The Molecular Basis of Cell Structure and Functionxe2x80x99, Albert L. Lehninger, Worth Publishers Inc. 1970, p 513-514 discloses that acetic acid can be a metabolic precursor of longer chain fatty acids in an isolated supernatant fraction of the liver following centrifugal removal of mitochondria, but only in the presence of carbon dioxide or bicarbonate. A metabolic pathway through malonyl CoA formed from acetyl CoA is described.
U.S. Pat. No. 5,378,612 describes low-protein media in which Pluronic F-68 surfactant is used in combination with cyclodextrin. Butyric acid is added to enhance protein expression and lithium salts, including lithium acetate, are also added for this purpose. No growth promoting effect due to the addition of carboxylic acids and their salts is noted or intended however. The media described are unsuitable for producing cell growth. They allow a short period of protein production prior to eventual cell death. This method of operation is necessitated by the lack of an adequate protein free growth medium. Several other disclosures have also noted the protein expression enhancing effect of butyric acid without describing any cell growth promoting effect.
Ethanol has been used in preparing culture media for the purpose of dissolving fatty materials including cholesterol and other hydrophobic compounds when making a concentrate to be diluted in aqueous media at a later stage, e.g. in WO-A-9204988. Accordingly, ethanol has been present in some cell culture media in the past in small quantities carried over from its use as a solvent for lipids such as cholesterol. For instance, CMRL 1066 (a standard basal medium formulationxe2x80x94see xe2x80x9cCulture of animal cellsxe2x80x9d R. I. Freshney, 3rd Edition, Willey-Liss), contains 16 mg/l ethanol for this reason. WO92/22637 describes solublising cholesterol in ethanol, followed by the production of liposomes for addition to culture media. The quantities of ethanol present in the cell culture media through this route will however not have been adequate to provide a significant growth promoting effect as described hereafter according to the invention. Also, when ethanol is included in a serum supplemented medium, no growth promoting effect is seen.
We have now made a number of discoveries resulting in improved cell culture media that may be employed separately or in combinations of some or all of such improvements.
First, we have now found surprisingly that surfactant, e.g. a surfactant of the type known as PLURONIC F68 (PF68) can be used to the exclusion of protein agents to incorporate otherwise water insoluble lipids or lipid precursors such as cholesterol and/or other sterols into serum-free media. PLURONIC F68 is also known under the names POLOXAMER 188, POLOXALKOL and EXOCORPOL.
This enables the lipid requirement to be satisfied by a stable, synthetically produced, chemically pure material, whereas in media such as CMRL 1066, the cholesterol would precipitate out and in more recent media the cholesterol is not present in pure form but stabilised by proteins which are purified from natural sources and introduce problems of lack of reproducibility and potential contamination.
In a first aspect, the present invention provides a cell culture medium containing one or more synthetic lipids or lipid precursors, e.g. a sterol or a metabolically acceptable derivative thereof in solution stabilised by one or more surfactants and in the substantial absence of protein and of phospholipid.
The cell culture media of the invention are preferably growth media.
The sterol or derivative thereof may be one or more of cholesterol, campesterol, desmosterol, ergosterol (produced by yeast), fucosterol, xcex2-sitosterol (produced by soya beans), stigmasterol, or a metabolically acceptable derivative thereof. It may be present as a said derivative which is an ester. Other metabolically acceptable derivatives may be used, i.e. derivatives which perform the role of such sterols in cell culture satisfactorily and without toxic effects. Suitably, the ester is an acetate. As further alternatives to cholesterol, other sterols including, other methyl cholesterols, various hydroxy-cholesterols, epi-cholesterol, cholesterol, and xcex2-estradiol may be used.
Having regard to the superior results described below, the invention includes a cell culture medium containing a mixture of campesterol and xcex2-sitosterol.
For use in all aspects of the invention, the sterol is preferably not a sterol produced by animals or is at least not a sterol produced by mammals. Sterols that are also produced by animals are preferably not used even if the actual source of the sterol used is non-mammalian. For instance one may use a plant sterol (being a sterol synthesised by plants but not by animals), a microbial sterol (being a sterol synthesised by a micro-organism (e.g. bacteria or yeast) but not by animals) or a non-natural sterol (i.e. a sterol made synthetically and not found in nature). Non-natural sterols include sterols produced by genetically engineered organisms and which are not found in nature but not sterols synthesised by any naturally occurring living organism.
Sterols that are synthesised by both plants and micro-organisms (but not animals, or not mammals) are not included amongst the sterols which may advantageously be used in the various aspects of the invention.
Thus, preferred sterols for each aspect of the invention exclude cholesterol (however actually produced).
It is a surprising finding of the inventors that culture media according to the invention containing non-mammalian or non-animal sterol can be used successfully or even advantageously in the culture, especially growth culture, of animal (including mammalian) cells, such as human cells.
The preferred sterols (i.e. the non-animal sterols) may be used in combination with one another and may also be used in combination with one or more animal sterols (whether naturally or synthetically produced). For instance, the non-animal sterols may be used in combination with cholesterol.
Mixtures of non-animal sterols that may be used include stigmasterol and xcex2-sitosterol, e.g. in a weight ratio of from 2:1 to 6:1.
There is further provided a cell culture medium containing cholesterol or another sterol having similar properties in cell culture solubilised by an xcex1-hydro-w-hydroxypoly oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymer surfactant of the structure HO(CH2CH2O)a(CH(CH3)xe2x80x94CH2OH)b(CH2CH2O)cH where a is from 50 to 100, b is from 20 to 40 and c is from 50 to 100, with an average molecular weight of from 7,500 to 10,000 in the substantial absence of other cholesterol solubility stabilising agents, i.e. proteins such as serum components or albumen.
Preferably, a=75, b=30, and c=75 and said average molecular weight is about 8350.
More preferably, said surfactant is PLURONIC F68. Other surfactants, particularly non-ionic surfactants that do not interfere with the action of the cell culture medium may be included but preferably are absent, e.g. polyoxyethylene sorbitan monooleate type surfactants such as Tween 80.
However, it has been found that the natural surfactant cholic aid may in certain types of cell culture provide a growth stimulating effects. Accordingly, the invention includes a cell culture medium containing cholic acid.
Preferably, the culture medium contains from 0.05 to 20 mg/l of cholesterol, more preferably from 0.5 to 10 mg/l, for instance from 0.05 to 2 mg/l.
Preferably, the culture medium comprises from 20 to 300 mg/l of said surfactant, more preferably 50 to 200 mg/l, more preferably about 100 mg/l.
The cholesterol or other sterol and the surfactant may be prepared in a concentrated solution for addition to a basal medium to form a culture medium containing these materials in the required quantities. Such a concentrate is conveniently prepared by dissolving them in alcohol, preferably ethanol, and the addition of such a concentrate to a basal medium as described in detail hereafter can provide to the basal medium a quantity of alcohol sufficient to provide a cell growth promoting effect according to an alternative aspect of the invention.
The cell culture medium may further contain a soluble carboxylic acid or metabolically acceptable derivative thereof as a metabolic precursor of lipid fatty acids. Said carboxylic acid is preferably a C2 to C7 carboxylic acid which is preferably saturated and preferably contains one carboxylic acid group. The carboxylic acid may be added as the acid as such or in the form of a metabolically acceptable salt or other derivative such as an ester. Preferably, said carboxylic acid or derivative is present at a concentration of from 10 xcexcM to 10 mM, more preferably from 50 xcexcM to 1 mM, e.g. from 100 to 300 xcexcM, most preferably at about 100 xcexcM.
In a further aspect, the invention provides a cell culture medium containing an alcohol capable of promoting cell growth at an effective growth promoting concentration of at least 0.01% vol/vol acting as a cell growth promoting agent.
Preferably, the said alcohol concentration is from 0.01% to 0.25% vol/vol, more preferably from 0.075% to 0.2% vol/vol, for instance from 0.1% to 0.15% vol/vol.
Said alcohol may be a C1 to C4 mono-alcohol or diol. Suitable alcohols include methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol or glycerol but ethanol is most preferred. Preferably the alcohol concentration is at least 0.01% to 0.25% vol/vol , e.g. from 0.075% to 0.2% vol/vol, more preferably from 0.1% to 0.15% vol/vol (1% ethanol vol/vol=7.9 g/l).
The invention includes in this aspect the use of an alcohol, especially ethanol to promote cell growth in culture.
As mentioned above cholesterol is included in some cell culture media to provide lipid, often in combination with other lipids such as lecithin, sphingomyelin, fat soluble vitamins (A, D, E and K), phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, dipalmitoyl phosphatidic acid and fatty acids. The incorporation of these hydrophobic materials poses problems. This has been addressed in the past by the use of complex lipoproteins, cyclodextrin or lipid micelles maintained by albumen to stabilise the cholesterol.
A further difficulty in preparing satisfactory serum-free media in the past has related to the incorporation of fatty acids. These pose a solubility problem and in addition are chemically unstable, being subject to oxidation. We have surprisingly found that soluble carboxylic acids and their derivatives such as salts and esters can be utilised in cell culture for the synthesis of fatty acids by cells, removing the need to include fatty acids in the culture medium itself. Certain basal media formulations include one or more fatty acids such as linoleic acid. However, in these formulations the linoleic acid is not present in a useful form. It will precipitate at once when the medium is made ready for use. It is therefore necessary in practice to add lipid solubilising materials such as serum, albumen or liposomes to make the media acceptable to the cells. Accordingly, according to a further aspect of the invention there is now provided a growth medium for cell culture comprising a soluble carboxylic acid or metabolically acceptable derivative thereof for providing a metabolic precursor of lipid fatty acids and/or for increasing cell growth rate. Suitable carboxylic acids and their derivatives are described above.
Preferably, the concentration of the soluble carboxylic acid or salt in the culture medium is given above.
The medium may comprise polyvinylpyrrolidone such as PVP-10, molecular weight 10,000. Suitably this is present at a concentration of from 25 to 10,000 mg/l, more preferably 100 to 500 mg/l, e.g. about 250 mg/l.
The culture medium may in addition contain all or any of the ingredients in the proportions or amounts disclosed in U.S. Pat. No. 5,045,454 for use in that invention.
As disclosed there, the medium preferably comprises one or more chelators preventing precipitation of iron at culture temperatures, typically 37xc2x0 C. The most preferred chelators are EDTA (ethylenediaminetetraacetic acid) and citrate buffer. Preferably the molar ratio of citrate buffer to EDTA is at least 3:1 citrate buffer: SDTA by molar concentration, more preferably about 10:1 citrate buffer: SDTA, where citrate buffer represents the sum of citric acid and citrate.
Iron is preferably supplied in the medium as the metal chelate complex defined as Fe-EDTA together with citrate buffer. More particularly, there may be provided about 2.4 to 3.6 xcexcM Fe-EDTA and about 0.8 to 1.2 xcexcM Na2(or K2) EDTA with about 12 to 18 xcexcM citric acid and about 20 to 30 xcexcM Na3 Citrate. Other biocompatible chelating agents may be used.
An agent for presenting iron to the cells is preferably provided which preferably is aurintricarboxylic acid. This is preferably present at a concentration of from 0.1 to 10 xcexcM, more preferably from 2.4 to 3.6 xcexcM, e.g. 3 xcexcM.
The pH of the medium may be between about 7.0 and 7.8, most preferably about 7.4 but the optimum may vary according to the nature of the cells in culture.
The culture medium is preferably serum-free and also protein-free except that it may be advantageous to add a small quantity of insulin, as described in U.S. Pat. No. 5,045,454.
Media containing insulin at the appropriate concentration for its use may still be regarded as substantially protein free.
Analogous to the methods described there, the culture media according to the various aspects of the present invention may be prepared by making up one of a number of standard or modified basal media and adding to it the additives disclosed in U.S. Pat. No. 5,045,454 from one or more concentrate solutions and the further additives required by the present invention from one or more further concentrates.
The basal media may be any of those known in the art including Eagle""s MEM, Dulbecco""s modified Eagle""s MEM, Ham""s F10/F12, CMRL, RPMI 1640, 199, L15, Fischer""s or Waymouth""s MB 752/1 (see Ref. 1). Preferably however, the basal medium is RPMI 1640. RPMI-X (a modified RPMI 1640 containing 20 mM HEPES and pyruvate) is preferred for the culture of cells of the leukocyte and reticulo-endothelial lineages, including lymphocyte hybridomas, while DME/F12 is preferred for use for other types of cells.
The recommended concentration of bicarbonate is 2.2 g/l and antibiotics such as penicillin or streptomycin should preferably not exceed 50 U/ml and 50 xcexcg/ml respectively.
To such basal media may be added an appropriate amount of a xe2x80x9cSSR2xe2x80x9d as described in U.S. Pat. No. 5,045,454, i.e. a solution containing EDTA, citrate, iron and aurintri-carboxylic acid as well as optionally PLURONIC F68, e.g. a xe2x80x9csolution Axe2x80x9d (U.S. Pat. No. 5,045,454, Col. 5) containing 20 mg/ml PLURONIC F68, about 4 mM EDTA, about 3 mM Fe, about 40 mM sodium citrate/citric acid, about 3 mM aurintricarboxylic acid and optionally about 1% trace elements. These amounts are suitable for a 1000xc3x97 formulation, i.e. a solution containing 1000 times the concentration of each ingredient desired in the final solution and which is therefore intended to be diluted with 1000 parts of the basal culture medium. The trace elements may comprise Mn (about 1 xcexcM), Cr (about 1 xcexcM), Zn (about 0.1 xcexcM), Ni (about 0.2 xcexcM), Co (about 0.2 xcexcM), Cu (about 20 xcexcM), Al (about 2 xcexcM) and Se (about 10 xcexcM).
The basal culture medium may be further supplemented with an appropriate quantity of a further concentrate solution of the kind referred to as xe2x80x9cSolution Bxe2x80x9d in U.S. Pat. No. 5,045,454 comprising about 0.5 mg/ml insulin (1000xc3x97).
Having regard to the above, the invention includes an additive for addition to a basal cell culture medium to form a serum-free cell culture medium, comprising:
an alcohol
a sterol or metabolically acceptable derivative
thereof, and
a soluble carboxylic acid.
The invention further includes an additive for addition to a basal cell culture medium to form a serum-free cell culture medium, comprising equal volumes of 96% ethanol and water and 100 g/l Pluronic F68.
To put into effect the various aspects of the present invention, the resulting supplemented basal medium may be further modified by the addition of an appropriate quantity of a further concentrate comprising cholesterol in ethanol or methanol solution, optionally also containing acetic acid. Other ingredients that may be included here are PVP 10 and ethanolamine.
A suitable composition for such a 1000xc3x97 xe2x80x9cSolution Cxxe2x80x9d will be:
ethanol 96% 1 liter
cholesterol (cell culture tested) 2 g (e.g. Sigma C-7402 C-3405)
PVP-10 250 g (plant cell tested) e.g. Sigma P-2307 concentrated acetic acid 6 ml (to 100 mM)
1.2 ml concentrated solution of ethanolamine (to 20 mM)
pH of concentrate 6.5
Depending on its intended use, the cell culture medium may be further supplemented using a concentrate xe2x80x9cSolution Dxc3x97xe2x80x9d containing ethanol and PVP-10, e.g. a 1000xc3x97 solution containing:
ethanol 96% 500 ml
water 500 ml
PLURONIC F68 100 g (Serva 35724 or Sigma P-1300)
We have found that certain commercially available basal culture media are capable of being improved by an iron conditioning process which results in a growth stimulating effect that may be due to the chemical reduction of organic molecules and the absorption of toxic contaminants into a fresh precipitate of iron hydroxide produced by the process.
Accordingly, the invention includes in a further aspect a method of conditioning a basal culture medium for increased cell growth comprising including in water used for making up a powder basal cell culture medium acidified ferrous sulphate and precipitating iron hydroxide from the made up powder by the addition of bicarbonate.
The conditioned basal medium so produced should be sterile filtered to remove the precipitate prior to the addition of solutions A, B, Cx and, if required, Dx described above, if the other aspects of the invention are to be used also.
A 1000xc3x97 solution for use in said conditioning step may be prepared by dissolving FeSO4 to 50 mM in 100 ml water plus 0.2 ml 2.5 M H2SO4 and diluting by 10xc3x97 in water.
Commercially available media powders produced by Sigma and Imperial benefit from this conditioning process. Generally speaking, the conditioning process will not be appropriate for conventional IVF media and Ham""s media, HF 10/12, MCDB media and DME/F 12 contain relatively high amounts of unstable ferrous sulphate and should not be treated in this way. The conditioning process should therefore in general be applied to basal media that will as a result produce better cell growth.